Recording Synaptic Currents at the Drosophila Larval Neuromuscular Junction

Place a Petri dish containing a dissected Drosophila larva with exposed muscles and nerve branches onto the microscope stage.

Insert a reference electrode into a bath solution within the Petri dish.

Fill a recording electrode with an isotonic solution.

Using a micromanipulator, position this electrode over an abdominal muscle innervated by motor neurons.

Place the electrode tip on a synaptic bouton, a structure at the axon tip containing neurotransmitters.

Continuously record miniature excitatory junctional currents or mEJCs, which are responses to spontaneous neurotransmitter release.

The correct range of mEJC amplitude confirms the proper electrode positioning.

Next, position a stimulation electrode near the axon that innervates the targeted muscle.

Apply negative pressure to pull the axon inside the electrode.

Gradually increase the stimulation current to induce an action potential.

This triggers the axon to release neurotransmitters at the neuromuscular junction, which are recorded as synaptic currents.

Place the petri dish with the preparation on the microscope stage. Insert the reference electrode in the bath. Fill the recording electrode with HL3 solution. Under the 10x objective, immerse the electrode into the bath, and place it over muscles six and seven of the abdominal segments 2, 3, or 4 using the micro-manipulator.

Next, switch the objective to 60X and focus on the area of interest using either epi-florescence or DIC optics. Place the tip of the electrode on top of the synaptic boutons, then, press the electrode very gently onto the muscle as excessive pressure may damage the NMJ or induce an increase in spontaneous synaptic activity.

Next, switch on the amplifier, AD board, and the computer. Then, choose the voltage clamp mode on the amplifier. Start the acquisition software, and choose the Gap-free mode, and observe the appearance of mEJCs on the computer screen. Ensure that the amplitude of the mEJCs is in the range of 0.2 to 0.7 nanoamperes.

Using a micro-manipulator under a visual control, place the stimulation electrode near the axon innervating abdominal segments 2 through 4. Apply negative pressure by pulling the piston of the syringe connected to the electrode holder, so that the axon is pulled inside of the electrode.

Next turn on the stimulator. Turn the knob on the isolation unit to set a zero current, and then, gently increase it until the EJCs appear, or until the threshold is reached. Perform the stimulation in a suprathreshold regime, with the stimulation current increased approximately twice compared to the threshold for the observation of EJCs.